Nitrogen fixation in pine: Direct confirmation and location of the diazotrophic population

氮通常是自然生态系统中最具限制性的植物营养素，这使得能够通过生物固氮（BNF）提供自身供应的物种具有明显的优势。植物伴生生物固氮可以满足某些非固氮草本植物的部分氮素需求，但目前已知的非固氮草本植物是唯一能从生物固氮中获得大部分氮素的植物甘蔗生物固氮的病原菌最初被认为是一种重氮营养型葡萄糖酸醋酸杆菌（Gluconoacetobacter diazotrophicus）的内生菌株，但最近的研究表明，固氮营养的主要来源可能涉及一群细菌，可能包括生活在植物组织上或内部的未培养菌株。根据报告，在甘蔗和其他草本植物物种的生物固氮的微生物是可培养的内生菌，我寻找类似的微生物在商业上重要的裸子植物，lodgepole松和分离出几个可培养的多粘类芽孢杆菌菌株，具有显着的乙炔还原活性，从表面消毒lodgepole松幼苗和树组织的提取物。其中一个菌株P2b-2 R处理的松树幼苗，从BNF中获得高达79%的叶氮，并积累了显着高于对照的生物量。到目前为止，我的研究结果表明，（i）松树幼苗可以获得几乎所有的叶片氮固氮P2b-2 R，（ii）生物固氮P2b-2 R导致幼苗生长促进，如果幼苗生长足够长的时间和（iii）P2b-2 R的内生习性促进生物固氮。然而，迄今为止进行的实验本质上是探索性的，没有直接测量N2固定或排除促进幼苗生长的其他可能原因。我建议确认我们的间接证据的BNF P2b-2 R在松树中使用15 N组织掺入试验，并确定是否松幼苗生长促进这种微生物的结果从BNF使用P2b-2 R nif-衍生物的野生型。此外，由于P2B-2 R殖民内部和外部根，茎和针叶组织，我建议确定这些植物相关的N2固定人口的贡献最大的松树相关的生物固氮。

Nitrogen is often the most limiting plant nutrient in natural ecosytems rendering a distinct advantage to species able to provide their own supply through biological nitrogen fixation (BNF). Plant-associated BNF can satisfy part of the plant N requirement in certain non-nodulating herbaceous species but the only known non-nodulating plants that can obtain most plant N from BNF (up to 80%) comprise a few cultivars of sugarcane.The causative agent of BNF in sugarcane was initially thought to be an endophytic strain of Gluconoacetobacter diazotrophicus, but more recent studies indicate that the primary source of diazotrophy may involve a consortium of bacteria, possibly including uncultured strains, that live on or inside plant tissues. Based on reports than the microorganism responsible for BNF in sugarcane and other herbaceous plant species were culturable endophytes, I searched for similar microorganisms in the commercially important gymnosperm, lodgepole pine and isolated several culturable Paenibacillus polymyxa strains that possessed significant acetylene reduction activity from extracts of surface-sterilized lodgepole pine seedling and tree tissues. Pine seedlngs treated with one of these strains, P2b-2R, derived up to 79% of foliar N from BNF and accumulated significantly more biomass than controls. My results so far suggest that (i) pine seedlings can derive almost all foliar N from nitrogen fixation by P2b-2R, (ii) BNF by P2b-2R causes seedling growth promotion, if seedlings are grown for a sufficiently long time and (iii) P2b-2R's endophytic habit facilitates BNF. However, the experiments conducted so far were exploratory in nature and did not measure N2 fixation directly or rule out other possible causes of seedling growth promotion. I propose to confirm our indirect evidence of BNF by P2b-2R in pine using a 15N tissue incorporation assay and to determine if pine seedling growth promotion by this microorganism results from BNF using a P2b-2R nif- derivative of the wild type. In addition, because P2b-2R colonizes internal and external root, stem and needle tissues, I propose to determine which of these plant-associated N2 fixing populations contributes most to pine-associated BNF.